As used herein, various symbols have the meaning assigned in the ensuing table:
Ala=alanine PA1 Arg=arginine PA1 Asp=aspartic acid PA1 Boc=t-butyloxycarbonyl PA1 Cpc=cyclopentane carbonyl PA1 Cys=cysteine PA1 Glu=glutamic acid PA1 &lt;Glu=pyro-L-glutamic acid PA1 Gly=glycine PA1 Hip=Hippuric acid (Benzoyl-glycine) PA1 His=histidine PA1 Ile=isoleucine PA1 Leu=leucine PA1 Lys=lysine PA1 Met=methionane PA1 Orn=ornithine PA1 Phe=phenylalanine PA1 Pro=proline PA1 .DELTA.Pro=3,4-dehydroproline PA1 Ser=serine PA1 Thr=threonine PA1 Thy=thyronine PA1 Trp=tryptophan PA1 Tyr=tyrosine PA1 Val=valine PA1 ACE=angiotensin converting enzyme PA1 Hepes=N-2-hydroxyethylpiperazine-N'-2-enthanesulfonic acid PA1 R is hydrogen or an acyl group such as formyl, acetyl, propanoyl, butanoyl, phenylacetyl, phenylpropanoyl, benzoyl, cyclopentane-carbonyl, tert-butyloxycarbonyl, cyclopentanecarbonyl-L-lysyl, L-arginyl, L-lysyl or pyro-L-glutamyl. PA1 A.sub.1 is (i) a residue of a compound having at least one carboxyl group and at least one alpha- or beta-amino or alpha-imino group, such as phenylalanine, alanine, tryptophan, tyrosin, isoleucine, leucine, histidine, valine, glycine, phenylglycine, beta-benzylaspartic acid, gamma-benzyl glutamic acid, S-benzyl-cysteine, O-benzyl-serine, O-benzyl tyrosine, O-benzyl threonine, betaphenyl serine, thyronine, beta-2-thienylserine, beta-2-thienylalanine, alpha-methyl-histidine, alpha-methyl tyrosine, alpha-methyl phenylalanine, alpha-methyl tryptophan, tyrosine having a halo, nitro, methoxy or hydroxy substitutent, phenylalanine having a halo, nitro, amino or methoxy substituent, tryptophan having a fluoro, methyl or methoxy substituent, methionine, cysteine, arginine, omega-nitro-arginine, lysine, ornithine, aspartic acid, asparagine, glutamic acid, glutamine, homocysteine, penicillamine, norleucine, serine, beta-alanine, ethionine, homoserine, isoserine, norvaline, threonine, alpha-aminobutyric acid, alpha-aminoisobutyric acid, beta-cyclohexanyl-alanine, O-phosphothreonine, S-ethylcysteine, vinyl glycine, the alpha-methyl derivative of any of valine, leucine, isoleucine, cysteine, methionine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, lysine and arginine; proline alpha-methyl proline, 3,4-dehydroproline, thiazolidine-4-carboxylic acid, cycloleucine, pyroglutamic acid, 1-amino-1-cyclopropane-carboxylic acid, 1-amino-1-cyclobutane carboxylic acid, 1-amino-1 cyclohexane carboxylic acid, or proline having a halo or hydroxy substituent; (ii) is in amide or imide linkage with R.sub.1 when R.sub.1 is acyl, and (iii) is in thioester linkage through a carboxyl group with -S-. PA1 S is a sulfur atom; and Z represents the grouping EQU -B-A.sub.2 PA1 B is selected from, e.g.
Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I to an octapeptide, angiotensin II by removal of the carboxy-terminal. Angiotension I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, on a serum alpha-2-globulin.
Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPheArg is a powerful depressor (blood pressure lowering) agent. In addition to a direct pressor effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentrations in the blood of patients with renal hypertension.
The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors [Gavras, I., et al, New Engl. J. Med. 291, 817 (1974)].
ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked alpha-carboxyl group. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl-terminal end yielding as reaction products a dipeptide and a remnant.
The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied by proline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Biochemistry, 2nd Ed., Worth Publishers, Inc., New York, 1975, pp. 189-195. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of competitive substrates include bradykinin, and the peptide BPP.sub.5a (also called SQ20475) from snake venom, having the sequence &lt;GluLysTrpAlaPro.
Numerous synthetic peptide derivatives have been shown to be ACE inhibitors by Ondetti et al in U.S. Pat. No. 3,832,337 issued Aug. 27, 1974.
The role of ACE in the pathogenesis of hypertension has prompted a search for inhibitors of the enzyme that could act as antihypertensive drugs. See, for example, U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511 and 4,053,651. A highly effective inhibitor, with high biological activity when orally administered, is D-3-mercapto-2-methylpropanoyl-L-proline, designated SQ 14225, captopril, or capoten, disclosed in U.S. Pat. No. 4,046,889 to Ondetti et al, issued Sept. 6, 1977, and in scientific articles by Cushman, D. W. et al, Biochemistry 16, 5484 (1977), and by Ondetti, M. et al, Science 196, 441 (1977). The inhibitor SQ 14225 reportedly has an I.sub.50 value of 2.3.times.10.sup.-8 M. The I.sub.50 value reported by Cushman, et al, supra, is the concentration of inhibitor required to produce 50% inhibition of the enzyme under a standard assay system containing substrate at a level substantially above K.sub.m. It will be understood that I.sub.50 values are directly comparable when all potential factors affecting the reaction are kept constant. These factors include the source of enzyme, its purity, the substrate used and its concentration, and the composition of the assay buffer. All I.sub.50 data reported herein have been performed with the same assay system and same enzyme (human urinary ACE) and with an approximately 1/2K.sub.m level of substrate and are therefore internally consistent. Discrepancies with data obtained by other workers may be observed. Indeed, such discrepancies do exist in the literature, for unknown reasons. See, for example, the I.sub.50 values for BPP.sub.9a reported by Cushman, D. W. et al, Experientia 29, 1032 (1973) and by Dorer, F. E. et al, Biochim. Biophys. Acta 429, 220 (1976).
The mode of action of SQ 14225 has been based upon a model of the active site of ACE developed by analogy with the better known related enzyme, carboxypeptidase A. The active site was postulated to have a cationic site for binding the carboxyl end group of the substrate and a pocket or cleft capable of binding the side chain of the C-terminal amino acid and providing especially tight binding for the heterocyclic ring of a terminal proline residue. A similar pocket for the penultimate amino acid residue was postulated, and the published data suggested a rather stringent steric requirement, since the D-form of the inhibitor was substantially more potent than its stereoisomer or the 3-methyl and unsubstituted analogs. The sulfhydryl group on the inhibitor, postulated to be bound at the active site near the catalytic center, was believed to play a central role in inactivation of the enzyme by combining with the zinc moiety known to be essential for catalytic activity. Substituents on the sulfhydryl, such as a methyl group, and an S-acetyl derivative, substantially reduced potency of the inhibitor. See Cushman, D. W., et al, Biochemistry, supra.
In vitro study of the mechanism by which SQ 14225 and its analogs act to inhibit ACE has been somewhat hampered by the instability of these molecules under ambient conditions. For example, it has been observed that a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ 14225 at a pH of about 8 becomes substantially less active upon standing for as little as 30 minutes, and that activity continues to decrease as the solution stands for longer periods. It is believed that this loss in activity is mainly the result of dimerization of SQ 14225 occurring at the sulfhydryl end groups, whereby a disulfide is formed which is largely inactive as an inhibitor. Since the free sulfhydryl group is highly reactive and may be readily oxidized to polar acidic moieties such as sulfone and sulfoxide groups, it may also be that the observed in vitro loss of activity of aqueous solutions of SQ 14225 on standing is in some part a consequence of one or more such oxidation reactions, with formation of a sulfone or sulfoxide which does not function effectively as an inhibitor for ACE.
Reports of SQ 14225 clinical testing, some of which refer to the compound under the name "Captopril", suggest that the product is sufficiently stable in the normal gastric and intestinal environments of most patients to be an effective inhibitor for ACE when administered orally. It is not yet clear, however, whether there may be a group of patients for which SQ 14225 is substantially ineffective. Because of the high reactivity of the free sulfhydryl group, SQ 14225 could readily form mixed disulfides with serum, cellular proteins, peptides or other free sulfhydryl group-containing substances in the gastric or intestinal environments, in addition to the possibility for dimer formation or oxidative degradation reactions. A mixed disulfide with protein may be antigenic and, indeed, occasional allergic reactions have been clinically observed. See Gavras et al, New England J. Med. 298, 991 (1978). Disulfides and oxidative degradation products of SQ 14225, if formed, may at best be expected to be largely ineffective as inhibitors. It may be postulated accordingly that dose reponse to SQ 14225 may vary with conditions of administration and among individual patients. Moreover, in at least some patients, unwanted side effects may occur and maintenance of an effective concentration of the inhibitor in the body may be difficult to control.
Thioester compounds generally are thought to be highly reactive in that the thioester linkage is readily hydrolyzable to a sulfhydryl moiety and a carboxylic moiety. Thioesters are accordingly often used as active ester intermediates for acylation under mild conditions. Such groups as, e.g., acetylthio have been used as blocking groups in the above cited Ondetti et al patents. Thioester intermediates are also postulated to occur in the biosynthesis of cyclic peptides such as tyrocidin or gramicidin S. See Lipmann, F. in Accounts Chem. Res. 6, 361 (1973).
Compounds related to SQ 14225 have been disclosed by Ondetti et al, U.S. Pat. Nos. 4,046,889, 4,052,511, 4,053,651, 4,113,715 and 4,154,840. Of interest are disclosed analogs of SQ 14225 having the five-membered heterocyclic ring of proline replaced by a four- or a six-membered ring. The inhibitory potencies of such analogs relative to SQ 14225 are not disclosed. Substitution of D-proline for L-proline is reported to drastically reduce inhibitory potency of 3-mercaptopropanoyl amino acids (Cushman, D. W. et al, supra).